1. Field of the Invention
The present invention relates to a rapid method for the detection of ischemic states and to a kit for use in such a method. More particularly, the invention relates to the measurement of protein bound thiol (SH) groups to determine the presence or absence of ischemia.
2. Discussion of the Background
Progressive coronary artery disease may be well advanced without significant clinical symptoms such as chest pain or dyspnea. The sudden occlusion of a branch of a coronary artery resulting in a myocardial infarction (MI) dramatically signals the presence of long standing arterial wall disease such as calcification of the intima and wall, as well as progressive stenosis of the lumen of the artery.
Immediately following an ischemic heart event, proteins are released into the blood. Well known proteins released after an ischemic heart event include creatine kinase (CK), serum glutamic oxalacetic transaminase (SGOT) and lactic dehydrogenase (LDH). One well known method of evaluating the occurrence of past ischemic heart events is the detection of these proteins in a patient's blood. U.S. Pat. No. 4,492,753 relates to a similar method of assessing the risk of future ischemic heart events. Injured heart tissue releases proteins to the bloodstream after both ischemic and non-ischemic events.
Patients undergoing non-cardiac surgery may experience perioperative ischemia. Electrocardiograms of these patients show ST-segment shifts with an ischemic cause which are highly correlated with the incidence of postoperative adverse cardiac events. However, ST-segment shifts also occur in the absence of ischemia and, therefore, this method does not distinguish ischemic from non-ischemic events.
Ischemia is frequently caused by arterial vessel disease. One feature of arterial vessel disease is the progression from the atheromatous state to the sclerotic state in which large quantities of calcium enter the arterial musculature. With the passage of time, arteriosclerosis progresses. The quantity of intracellular calcium increases while cardiac output remains essentially normal. The intracellular calcium activates the protease calpain which converts xanthine dehydrogenase to xanthine oxidase. Xanthine oxidase acts on xanthine and hypoxanthine to form free radicals, including the hydroxyl radical (OH.) and the superoxide radical (O.sub.2.). These free radicals in turn oxidize cell membranes and proteins in the regions of the molecule which are rich in thiol groups. See "The Role of Perfusion--Induced Injury in the Pathogenesis of the Crush Syndrome", New Engl. J. Med., 324:1417-1422 (1991).
A need exists for a method of distinguishing between ischemic and non-ischemic events, particularly in cardiac patients. After substantial research, the present method, based on metal-protein binding interactions, has been discovered which is capable of detecting ischemic states or events in a patient.
It is well known that metal ions are capable of binding to metal-binding groups in proteins ("Multiple Equilibria in Proteins", J. Steinhardt and J. Reynolds, Acad. Press, CH-VI, p 214 et seq.). Metal ions may form covalent linkages with proteins or, alternatively, form coordination complexes where the metal ion is chelated by ligands of the protein molecule (Enzyme and Metabolic Inhibitors, Vol II. J. L. Webb, (1966), Acad. Press, Chapt. 4, page 635 et seq.).
The ability of metal ions to bind proteins forms the basis of silver stains for proteins in polyacrylamide gels. U.S. Pat. No. 4,468,466 pretreats a gel with dithiothreitol (DTT) prior to staining with silver ions to reduce background staining. U.S. Pat. No. 4,434,234 provides a subsequent treatment with carbonate or sulfate salts to obtain different color stains.
In some instances, metal ions react with proteins to form precipitates. Metal-protein precipitation reactions have been used in methods for the quantitative determination of protein (U.S. Pat. No. 4,786,605) and in the total or fractional precipitation of proteins from a protein-containing solution (U.S. Pat. No. 4,486,282).